Centrosomes are the major microtubule organizing centers (MTOC) in cells. Importantly, as poles of the mitotic spindle, centrosomes nucleate the microtubules responsible for proper chromosome segregation to the progeny cells. Failure in centrosome duplication or function leads to genomic instability and contributes to cellular transformation. Therefore, a detailed mechanistic understanding of duplication is critical in assessing how errors in this process result in centrosomal defects and potentially lead to a disease state. Using the genetically tractable budding yeast, Saccharomyces cerevisiae, in which the centrosome, known as the spindle pole body (SPB), is well defined, we propose to investigate the assembly mechanisms that produce new centrosomes, how phosphorylation regulates events at the centrosome, and whether specific regulatory phosphorylations are conserved among eukaryotes. We, and others, have previously shown that protein phosphorylation plays a significant role in the assembly and function of centrosomes. Recently, we isolated intact yeast SPBs and subjected them to mass spectrometric analysis, yielding a phosphoproteome that includes 297 mapped phosphorylation sites across 17 of the 18 core SPB components. We will use this novel resource to discover which phosphorylation events are important for SPB assembly and microtubule nucleation. In the first aim, we will test a model for the initiation of yeast SPB duplication based on the hypothesis that phosphorylation of the conserved SPB component Sfi1 regulates both its step-wise addition to the existing SPB and its ability to recruit SPB components that form the nascent SPB. In addition, we will test whether Sfi1 and its binding partner Cdc31 (centrin) alone are sufficient for initiation of SPB assembly. The second aim focuses on the expansion of SPBs in mitotically-arrested cells, as this expansion is similar to centrosome maturation. In both cases, components that are conserved between SPBs and centrosomes are added to the structures, and centrosome microtubule nucleation capacities increase. We will determine which SPB components, phosphorylation events, and regulators contribute to the expansion of SPBs in cdc20- depleted cells. This work will reveal core SPB assembly mechanisms and their regulation. The third and final aim focuses on the role of phosphorylation in the assembly and function of the 3-tubulin complex, consisting of three conserved proteins (Tub4, Spc97, Spc98) responsible for microtubule nucleation. Another important protein, Spc110, attaches the 3-tubulin complex to the SPB, and we will determine whether Spc110 phosphorylation is critical for 3-tubulin complex assembly and/or microtubule nucleation. Finally, we will ask whether conserved residues within the human 3-tubulin complex are also phosphorylated and function in a manner similar to those in the yeast 3-tubulin complex. In summary, our completed yeast SPB phosphoproteome allows us to move beyond the mapping of specific phosphorylation sites to the more complex questions of how phospho-regulation acts in centrosome assembly, structure and function. PUBLIC HEALTH RELEVANCE: The aim of this work is to understand processes regulating the assembly and function of the yeast centrosome. The centrosome forms the microtubules in the mitotic spindle. As such, centrosome duplication is a key cell cycle event producing the two poles of the spindle. Defects in centrosome structure and number are commonly observed in tumor cells, and contribute to the genetic instability in these cells. In order to understand this aspect of tumor cells, a complete understanding of centrosome assembly is required and our work in yeast will contribute to that understanding.